- Iranian Journal of Pharmaceutical Research

Iranian Journal of Pharmaceutical Research (2011), 10 (1): 63-68
Received: January 2009
Accepted: July 2009
Copyright © 2011 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services
Original Article
Hepatoprotective Effect of Ficus carica Leaf Extract on
Mice Intoxicated with Carbon Tetrachloride
Nasrin Aghel*, Heibatollah Kalantari and Shohreh Rezazadeh
Medicinal Plant Research Center, Joundishapour Medical Sciences University, Ahwaz,
Iran.
Abstract
Protective action of Ficus carica leaf ethanolic extract (obtained by maceration) was
evaluated in an animal model of hepatotoxicity induced by carbon tetrachloride (CCl4).
Male albino mice were divided into six groups. group I was normal control group; group
II received olive oil (CCl4 solvent), groups III-VI received CCl4. After inducing hepatic
damage, group III served as control for CCl4; and groups IV- VI received different doses of
Ficus carica ethanol extract (200, 400 and 800 mg/kg) prior to intoxication with CCl4. Liver
marker enzymes were assayed in serum. Sections of livers were observed under microscope
for the histopathological changes. Levels of marker enzymes such as alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) were increased significantly in CCl4 treated mice
(group III). In groups IV, V and VI, pre-treated with the plant extract and intoxicated with CCl4,
decreased activities of these two enzymes were observed. Also, pre-treatment with the extract
in these groups resulted in less pronounced destruction of the liver architecture with no fibrosis
and moderate inflammation was observed compared with group III. The present observations
suggested that the treatment with Ficus carica leaf extract in dose of 200 mg/kg enhanced
protection against CCl4 induced hepatic damage.
Keywords: Ficus carica; Liver; Carbon tetrachloride; Hepatoprotective; Mice.
Introduction
The genus Ficus belongs to the family
Moraceae and to the order urticales (1).
Moraceae is composed of trees and shrubs
which characteristically have a milky juice.
Ficus constitute one of the largest genera
of angiosperms, with almost 800 species of
terrestrial trees, shrubs, hemi-epiphytes, climbers
and creepers occurring in the tropics and
subtropics worldwide (2). Ficus are important
genetic resources with high economic and
nutritional value. They are also an important part
* Corresponding author:
E-mail: [email protected]
of the biodiversity in the rainforest ecosystem by
setting fruit throughout the year and providing an
important source of food for fruit-eating animals
in the tropics.
Ficus carica (fig tree) has been extensively
investigated for its proteolytic enzymes (3),
amino acids, minerals and sugars (4), triterpenes
(5), and organic acids (6). The leaves are added
to boiling water and used as a steam bath for
painful or swollen piles, and as a decoction
and stomachic (7). The fruit is mildly laxative,
demulcent, digestive and pectoral (8). The leaf
decoction is taken as a remedy for diabetes and
calcifications in the kidneys and liver (9).
One of the traditional methods for treatment
of warts in some rural areas of Iran is to use fig
Aghel N et al. / IJPR (2011), 10 (1): 63-68
from the leaves of Ficus carica against hepatic
injury induced by CCl4 in mice to determine the
possible use of this plant in preventing hepatic
damage.
tree latex as a local treatment, as reported by
Avicenna in his 10th century book Canon of
Medicine (10)
Despite the fact that other parts of the fig
tree, like the fig leaves, have also reported
pharmacological properties, they have been
much less investigated. In 1998, Serraclara et
al. (11) reported the hypoglycemic action of
a fig leaf decoction in type-I diabetic patients,
and in 2000, Canal et al. (12) used a chloroform
extract, obtained also from a decoction of F.
carica leaves, to decrease the cholesterol levels
of rats with diabetes. These pharmacological
properties are probably in part due to the high
content of phenolic compounds in these plant
extracts. Administration of the F. deltoidea
leaves aqueous extract produced significant
dose-dependent antinociceptive effect in animal
model (13).
Liver is the first major organ to be exposed to
ingested toxins due to its portal blood supply and
toxins may be, at least partially, removed from
the circulation during the first pass, providing
protection to other organs while increasing the
likelihood of hepatic injury (14). Liver toxicity is
monitored in standard toxicity studies by a range
of investigations including clinical biochemistry
parameters (enzymes, proteins, lipids, etc.).
The following endpoints are considered to
be mainly related to liver toxicity: its relative
weight and more than two enzymes indicative
of hepatocellular effects such as (alanine
aminotransferase, aspartate aminotransferase,
alkaline phosphatase and lactate dehydrogenase)
(15).
Unfortunately, conventional or synthetic
drugs used in the treatment of liver diseases
are inadequate and sometimes can have serious
side effects. This is one of the reasons for many
people in the world over including those in
developed countries turning complimentary and
alternative medicine. Many traditional remedies
employ herbal drugs for the treatment of liver
ailments (16). A number of plants have been
shown to possess hepatoprotective property
(17-19).
Considering that other species of Ficus
had hepatoprotective activity, the present
investigation was undertaken to test the efficacy
of different doses of ethanolic extract prepared
Experimental
Materials and methods
Animals
Male albino mice weighing 30-31.5 g
were obtained from Animal House of Ahwaz
Joundishapour University of Medical Sciences
and were kept at 23-25°C, under the light and
dark cycles of 12 h, at 60-80% relative humidity,
and fed with a standard diet and watered ad
libidum. All experiments were performed in the
morning.
Chemicals
Assay kits for the estimation of serum
enzymes (ALT and AST) were purchased from
pars azmun (liquid test). All other chemicals
were of analytical grade.
Plant material
Leaves of Ficus carica were collected
from campus garden of School of Pharmacy
in summer of 2007. The plant material was
identified and authenticated taxonomically by Dr.
Sedigi, Faculty of Agriculture, Shahid Chamran
University, Ahwaz, Iran and a voucher specimen
was stored in the herbarium of Pharmacognosy
Department, School of Pharmacy, Ahwaz
Joundishapour University of Medical Sciences,
Ahwaz, Iran.
Preparation of plant extract
The leaves were shade dried, powdered
and then were extracted with 80% aqueous
EtOH by maceration at room temperature for
72 h. The extract was filtered and the filtrate
was concentrated in a rotary evaporator under
reduced pressure.
CCl4 induced hepatotoxicity
Mice were divided into six groups of seven
animals in each group. Table 1 shows the dose
schedule of carbon tetrachloride and test samples
against CCl4 intoxication. Each mouse was
treated for four days with saline (0.9%), olive
64
Hepatoprotective Effect of Ficus carica Leaf Extract on Mice
Table 1. Dose schedule.
Groups
(I) Negative Control
Days
1
2
3
4
5
Normal Saline (0.9٪)
Normal Saline (0.9٪)
Normal Saline (0.9٪)
Normal Saline (0.9٪)
Sacrifice
(II) Olive Oil
Normal Saline
Olive Oil
Olive Oil
Normal Saline
Sacrifice
(III) CCl4
Normal Saline
CCl4
CCl4
Normal Saline
Sacrifice
(IV) Test 1
200 mg/kg
200 mg/kg + CCl4
200 mg/kg + CCl4
200 mg/kg
Sacrifice
(V) Test 2
400 mg/kg
400 mg/kg + CCl4
400 mg/kg + CCl4
800 mg/kg
800 mg/kg + CCl4
800 mg/kg + CCl4
400 mg/kg
800 mg/kg
Sacrifice
(VI) Test 3
Sacrifice
Negative control received normal saline, Test 1, 2 and 3 received 200, 400 and 800 mg/kg of Ficus carica leaf extract, respectively.
oil (CCl4 solvent); CCl4 (0.2 mL/kg in olive oil)
or test samples (plant extract) orally in the total
volume of 0.2 mL. Mice in test groups, in the
second and third day, were given CCl4 half an
hour after the administration of the plant extract
dose. Liver weight changes, biochemical and
histopathological evaluation were undertaken on
the fifth day.
Animals were killed in the fifth day by using
chloroform, their blood was collected, allowed to
clot and serum was separated by centrifugation
at 3000 rpm for 15 min. Liver was dissected out
and used for histopathological studies.
were analyzed by analysis of variance (ANOVA)
followed by multiple comparisons using Tuky
test to compare all groups against control.
Results were considered statistically significant
at p < 0.05.
Results and Discussion
Liver is a major site for metabolism of
exogenous chemicals (pesticides, drugs, metals),
resulting in the formation of metabolites which
may be more or less toxic than the parent
compound (20). CCl4, an extensively studied
liver toxicant, and its metabolites such as
CCl3 radicals are known to be involved in the
pathogenesis of liver damage.
The yield (w/w) of the ethanolic extract was
6.36%. No mortality observed in any of the
groups. The results of hepatoprotective effects
of different Ficus carica leaf extracts on CCl4
intoxicated mice are shown in Table 2. There was
no significant changes in the activities of serum
ALT, AST and liver weight in group of olive oil
mice compared to negative control group (Table
2). The liver weights of the three Test groups
calculated at the end of the study had decreased
(statistically significant) when compared with
CCl4 induced group (Table 2). In the three test
groups the level of marker enzymes were found
retrieving towards normalcy.
The histopathological observation basically
support the results obtained from enzyme assays.
Histology study of liver from negative control
group showed a normal hepatic architecture
(Figure 1). In CCl4 induced group, severe
hepatotoxicity (massive fatty changes, necrosis,
Biochemical determination
The biochemical parameters (serum
enzymes): alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) were
assayed spectrophotometrically using a
commercially available assay kits according to
the manufacturer’s protocol.
Liver histopathological assessment
Liver sections taken immediately from
liver, fixed in 10٪ formalin, dehydrated in
gradual ethanol (50-100٪), cleared in xylene
and embedded in paraffin. Sections (4-5
µm thick) were prepared and then stained
with hematoxylin and eosin (H-E) dye for
photomicroscopic observation, including cell
necrosis, fatty change, hyaline degeneration,
ballooning degeneration, infiltration of Kupffer
cells and lymphocytes.
Statistical analysis
The data are expressed as mean ± SD. Data
65
induced hepatotoxicity in rats. Phytother. Res. (2000) 14: 457-9.
22- Mandel SC, Maity TK, Das J, Pal M and Saha BP. Hepatoprotective activity
of Ficus racemosa leaf extract on liver damage caused by carbon tetrachloride
in rats. Phytother. Res. (1999) 13: 430-2.
23- Krishma Mohan G, Pallavi E, Ravi Kumar B, Ramesh M and Venkatesh S.
Hepatoprotective activity of Ficus carica Linn. Leaf extract against carbon
tetrachloride-induced hepatotoxicity in rats. Daru (2007) 15: 162-166.
Figure 2. Light microphotographs of HE- stained sections (100X) of the formalin fi
Aghel N et al. / IJPR (2011), 10 (1): 63-68
liver of carbon tetrachloride control group showing severe hepatotoxicity.
A
A
B
B
Figure 1. Light microphotographs of HE- stained sections
(100X) of the formalin fixed liver of normal control group
showing normal hepatic architecture.
Figure 1. Light microphotographs of HE- stained sections (100X) of the formalin fixed
liver of normal control group showing normal hepatic architecture.
C
C
C
Figure
2. Light
microphotographs
of HE-sections
stained(100X)
sections
Figure
2. Light
microphotographs
of HE- stained
of the formalin fixed
(100X)
of the
formalin control
fixed liver
carbon
tetrachloride
liver
of carbon
tetrachloride
group of
showing
severe
hepatotoxicity.
control group showing severe hepatotoxicity.
A
ballooning degeneration and broad infiltration
of the lymphocytes and Kupffer cells around the
central vein) was seen (Figure 2). The histological
architectusre of liver sections of mice treated
with different Ficus carica leaf extracts showed
a more or less normal lobular pattern with a mild
degree of fatty change, necrosis and lymphocyte
infiltration almost comparable to the normal
control group (Figure 3).
In previous investigations , hepatoprotective
effect of leaf extracts of other species of Ficus
were
examined. Mandal et al. showed that
B
an oral dose of 400 mg/kg of Ficus hispida
methanolic leaf extract exhibited a significant
protective effect against paracetamol-induced
hepatotoxicity in rats (21). The hepatoprotective
activity of Ficus racemosa leaf extract on liver
Figure 3.
3. Light
Light microphotographs
microphotographs of
of HEHE- stained
stained sections
sections (100X)
(100X) of
of the
the formalin
formalin fixed
fixed l
Figure
Figure
Figure
Light pre-treated
microphotographs
of(200
HE-mg/kg,
stained
of Ficus
Ficus3.
carica
leaf3.extract
extract
pre-treated
groups: A
A
(200
mg/kg,
p.o.); BB (400
(400 mg/kg,
mg/kg, p.o.)
p.o.)
of
carica
leaf
groups:
p.o.);
sections
(100X)
of the
formalin
fixedor
liver
Ficuslobular
carica pattern.
leaf
C (800
(800 mg/kg,
mg/kg,
p.o.);
showing
more
or
lessof
normal
lobular
pattern.
C
p.o.);
showing
aa more
less
normal
extract pre-treated groups: A (200 mg/kg, p.o.); B (400 mg/kg,
p.o.) and C (800 mg/kg, p.o.); showing a more or less normal
lobular pattern.
damage caused by carbon tetrachloride in rats
was comparable to standard liver tonic (22).
In another study, carried out by Krishna
Mohan et al., the protective effect of methanolic
leaves extract of Ficus carica was reported
at a dose of 500 mg/kg (23). In the present
research, we tried three different doses and
obtained a significant liver protection at dose
of 200 mg/kg.
66
Hepatoprotective Effect of Ficus carica Leaf Extract on Mice
Table 2. Effect of different Ficus carica leaf extract on CCl4 induced rise in serum ALT, AST and changes in liver weight in mice.
Groups (n = 7)
Negative Control
ALT (IU/L) ( Mean ± SEM)
AST (IU/L) ( Mean ± SEM)
Liver weight (g) ( Mean ± SEM)
116.75 ± 1.60
138.15 ± 5.05
1.60± 0.035
Olive Oil
117.14 ± 2.99
145.28 ± 2.95
1.41 ± 0.025
CCl4
190.90 ± 1.35+
239.40 ± 2.84+
1.89 ± 0.079+
Test 1
85.57 ± 4.23*
194.71 ± 9.88*
1.22 ± 0.074*
Test 2
96.71 ± 2.99*
204.00 ± 14.86*
1.50 ± 0.084*
Test 3
132.43 ± 15.76*
224.00 ± 11.21
1.50 ± 0.081*
Negative control received normal saline, Test 1, 2 and 3 received 200, 400 and 800 mg/kg of Ficus carica leaf extract, respectively.
p* < 0.05 as compared with CCl4 induced group. p+ < 0.05 as compared with negative control group.
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